Journal of Advanced Laboratory Research in Biology
E-ISSN: 0976-7614
Volume 11, Issue 1, January 2020
PP 1-6
https://e-journal.sospublication.co.in
Research Article
Impact of different concentrations of Sodium chloride on the Root growth,
Cell division and Chromosomal abnormalities in the root tips of Allium cepa
Harem Othman Smail
Department of Biology, Faculty of Science and Health, Koya University, Koya KOY45, Kurdistan Region-F.R. Iraq.
Abstract: The experiment was conducted to study the inhibition of the root growth, cell division and cytotoxic
effects of NaCl on onion bulbs (Allium cepa). The onion bulbs were treated with different concentrations of NaCl
(0.06, 0.12, 0.24 and 0.48 molarity) for 72 hours in a glass beaker. The results, based on the different concentrations
and exposure time showed that the mitotic index and the average onion root growth rate decreased significantly
compared with the control. Treatment with 0.48 molarity NaCl concentrations has a less percentage of root growth
(12.9%) whereas treatment with distilled water (control) has maximum root growth (40.63%). The mitotic index of
control was (12%) while onion treated with NaCl was decreased to (9.11%) in 0.48 molarity. It was found that the
chromosomal aberrations increased as the concentration of the NaCl increased when compared to control. The
recorded chromosomal abnormalities were micronuclei, budding nuclei, unequal-sized nuclei, c-mitosis, anaphase
bridge, and chromosome stickiness. The results showed that the higher concentrations of NaCl have more impact on
the root growth, cell division and chromosomal abnormalities in the root tips of Allium cepa.
Keywords: Allium cepa, Root growth, Mitotic index, Chromosomal abnormalities, NaCl.
1. Introduction
The genus Allium is one of the world's biggest
flora. As per The Plant List (2020) there are 887 species
of Allium under family Amaryllidaceae found in the
world including both wild and cultivated plants. Allium
species are very important herbaceous plant, it is used
worldwide as spices and as vegetables. In early
classifications of angiosperms, Allium and related
genera have been placed in the Liliaceae family. In the
more recent and competent taxonomic treatment of
the Monocotyledons, they are recognized as the
distinct family Alliaceae close to the Amaryllidaceae
(Dahlgren et al., 1985).
Low water availability, high and low temperatures
and air pollution are serious threats to agriculture and
directly affect growth and yield in both arid and semiarid areas. In a similar way, salinity affects many
physiological characteristics, such as growth rate, water
relations, photosynthesis, ion homeostasis, senescence,
and yield elements (Negrão et al., 2017). Salinity
significantly inhibits plant productivity of several
horticultural crops. Sodium chloride salts cause most of
the salt stresses in nature. When plants experience
high salinity, they develop various coping
*Corresponding Author: Harem Othman Smail
E-mail: harem.othman@koyauniversity.org.
Phone No.: +964-7705047235.
mechanisms that allow them to tolerate, avoid, or
escape the stressor. Those responses were examined at
the morphological, anatomical, physiological and
cellular stages (Teerarak et al., 2009).
Classical cytogenetic techniques are usually used
to detect changes in chromosomal morphology. In 1928
Stadler describes the impact of physical and chemical
agents on chromosomes (Maluszynska & Juchimiuk,
2005). The effect of colchicine on root mitosis in
Allium cepa was first described by Levan (1938). The
nomenclature used for chromosomes morphology is
suggested by Levan et al., (1964). The Allium test
(Levan, 1938, 1949) is based on a chromosome analysis
of the meristem cells of the Allium cepa apical root
cells to determine the effect of genotoxic substances. In
addition, the Allium cepa system provides important
information for evaluating the clastogenic and/or
aneugenic impact of an agent on the genetic material
(Leme & Marin-Morales, 2009).
Onion (Allium cepa L.) root meristem cells are
highly sensitive to genetic damage by chemicals, and
the Allium test that involves the length of the root and
chromosome aberration (CA) measurements, proven to
be an effective model system for measuring the
environmental cytogenetic potential of pollutants. The
Allium test is an excellent indicator for analyzing
0000-0001-9867-4289.
Received on: 1 November 2019
Accepted on: 11 December 2019
Effects of NaCl on the root tips of Allium cepa
antiproliferative effects on plant medicinal extracts
(Firbas & Amon, 2014). Although favourable
cytological features make genus Allium species
attractive subjects for research, only about one-third of
chromosome numbers are identified and detailed
cytological information is very limited (Ramesh, 2015).
For decades, the Allium chromosomes have been
studied for their diversity in size, structure and number
(Awe & Akpan, 2017).
The diploid chromosome number of Allium cepa is
well established i.e., 2n = 2x = 16 (Okumuş and
Hassan, 2000; Kim et al., 2002; Mukherjee & Roy,
2012).
2. Material and Methods
2.1 Onion preparation
Small onion (Allium cepa) bulbs of the similar
uniform size, weighing about 10-13g, were denuded
and scraped by removing the loose outer scales so that
the root primordia are immersed in the specific distilled
water (control) and NaCl concentrations (0.06, 0.12,
0.24 and 0.48 molarity).
2.2 Experimental procedure
In each experiment the exposure time of the onion
bulbs was 72 hours at 22°C; they have been protected
from direct sunlight. In order to minimize the effect of
the daytime rhythms, the plants have been exposed to
artificial medium intensity light. In another version of
the experiment, the 5 onion bulbs had been placed
directly into the experimental NaCl solution of different
concentrations. The NaCl sample being studied has
been divided into three parts which were applied
successively to the roots of the onions for 24 hour
period. So the roots got a fresh bath of the NaCl
solution sample every 24 hours. The experiment was
done after 72 hours and analysis of macroscopic and
microscopic morphology of the tissue followed (Firbas
& Amon, 2014).
2.3 Macroscopic parameters
Macroscopic parameters include the length of the
root and other parameters, like as the normal root
shape, number, color and turgidity after 72 hours.
2.4 Root harvest and slide preparation
Root tips from the germinated seeds were cut 12cm long and placed in a small specimen glass bottle
and fixed in acetic alcohol at 4-6°C for 24 hours. The
root tips were washed twice for 10 minutes each with
ice-cold water and allowed to dry in a watch glass. In
the watch glass 1 N HCl solution preheated to 60°C
was added to the root tips for 5 minutes and the HCl
was discarded. Repeat the HCl treatment a second time.
Two root tips have been moved individually to clean
microscope slides and from the growing tip cut 2mm.
The tips have been kept and the remaining solution was
Harem Othman Smail
discarded. 1% methylene blue stain was applied to each
slide to cover the root tip for approximately 3 minutes.
A glass coverslip was placed on the root tip and gently
tapped with a pencil eraser to disperse the cells
uniformly to form a monolayer to make the scoring
process easier for normal and aberrant cells at various
phases of the cell cycle (Vellaikkannu et al., (2017).
2.5 Microscopic parameters
The cytogenetic analysis consisted of the mitotic
index, scoring of aberrant cells and the proportion of
mitotic phases. Mitotic index was calculated as the
percentage ratio of dividing cells and the total number
of scored cells. In the dividing cells of root tips, the
proportion of mitotic phases was scored. The
percentage of each type of aberrant cells, such as
budding nuclei, micronuclei, c-mitosis, unequal-sized
nuclei, chromosome stickiness, and anaphase bridge
was calculated in accordance with the methods
previously described (Gabara et al., 2006; Glińska et
al., 2007).
The slides were seen under the light microscope
(Olympus) using the 100X objective lens with oil
immersion. On one slide for each treatment, a total of
450 cells, classified into interphase or dividing cells
(prophase, metaphase, anaphase and telophase) were
scored. The mitotic index (MI) was expressed as the
number of dividing cells per 100 cells scored.
Mitotic index (MI) is calculated by the following
formulae:
Mitotic index (%) =
(1)
X 100
2.6 Statistical analysis
A one-way analysis of variance (ANOVA) with the
Tukey test was used to analyze the relationship between
different NaCl concentrations and chromosomal
abnormalities. p < 0.05 is considered statistically
significant.
Total chromosomal abnormality calculated by the
following formulae:
Total abnormality (%) =
X 100
(2)
3. Results and Discussion
In this experiment, roots of Allium cepa are treated
with different NaCl concentrations (0.06, 0.12, 0.24 and
0.48 molarity), distilled water as control and for
selected time periods (24, 48 and 72 hours). The growth
rate is different for each treated concentration and
control. The growth rate of root tips of onion treated
with distilled water (control) is 40.63% more than root
tips of onion treated with NaCl concentrations (0.06,
0.12, 0.24 and 0.48 molarity) after 72 hours. The
growth rate of root tips of onion treated with NaCl
concentration of 0.06, 0.12, 0.24 and 0.48 molarity are
J. Adv. Lab. Res. Biol. (E-ISSN: 0976-7614) - Volume 11│Issue 1│January 2020
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Effects of NaCl on the root tips of Allium cepa
35.86%, 21.05%, 15.15% and 12.9% respectively. Five
replications were performed for each concentration and
control to calculate mean root length with standard
error of the mean (SEM) at selected time periods (Table
1).
Salinity affects plants in various ways, including
osmotic effects, specific-ion toxicity and/or nutritional
disorders (Läuchli & Epstein, 1990). The degree to
which one mechanism affects the plant over the other
depends on a number of factors, including the species,
genotype, plant age, ionic strength and composition of
the salinizing solution and the organ concerned (Munns,
2002). Allium cepa was used as the experimental
material in this study because chromosomal aberration
observed in plants resembles the aberration produced in
mammalian cells. A positive correlation existed
between the aberrations induced by omnacortil in plant
root tip cells and in cultured mammalian cells. This
suggests that the root tip system of the plant can be
recognized as an effective first-tier assay system for this
study type (Alege & Ojomah, 2014).
Slide overview of each meristematic root tip for
control and concentrated group (0.06, 0.12, 0.24 and
0.48 molarity) at 40X magnification as shown in Fig. 1.
The effect of different NaCl concentrations on the
mitotic index of the examined root tips and the total
cells examined (450 cells), classified into interphase
and dividing cells (prophase, metaphase, anaphase, and
telophase) was shown in Table 2 and Fig. 2.
The percentage of mitotic index in the control
group (12%) is higher than other treated groups (0.06,
0.12, 0.24 and 0.48 molarity of NaCl). Lower mitotic
index (9.11%) is observed in the treatment with 0.48
molarity of NaCl. The mitotic index of other treated
concentrations is significantly different in comparison
with control. The percentage of mitotic index in 0.06,
0.12 and 0.24 molarity NaCl concentration is 11.55%,
11.33% and 10.88% respectively. Treatment with
control and different NaCl concentrations, there is a
significant difference between interphase and dividing
cells (prophase, metaphase, anaphase, and telophase).
Interphase has the highest number (409 cells) in the
0.48 molarity, while the control has the smallest
number (396 cells). For other groups, the number of
interphase cells was almost similar (0.06 molarity has
398 cells, 0.12 molarity has 399 cells and 0.24 molarity
has 401 cells) Table 2.
Among dividing cells prophase has a higher
number from metaphase, anaphase and telophase.
Individually 0.24 molarity NaCl concentration has the
highest number of prophase (33 cells) and 0.06 molarity
NaCl concentration has the smallest number of
prophase (24 cells). The other three dividing cells
(metaphase, anaphase, and telophase) also differ
between treated groups, 0.12 and 0.24 molarity NaCl
concentration has the same and the smallest number of
metaphase (4 cells only) while 0.06 molarity NaCl
concentration has the highest number of metaphase
Harem Othman Smail
cells (16) followed by control group (14 cells). The
lowest number of anaphase seen in 0.48 molarity NaCl
concentration (2 cells) followed by 0.06 molarity (6
cells), 0.24 molarity NaCl concentration and control
group has the same number (8 cells). The largest
number of anaphase has seen in 0.12 molarity NaCl
concentration (9 cells). In telophase 0.48 molarity NaCl
concentration has the lowest number (3 cells), 0.24
molarity NaCl concentration (4 cells) followed by the
control group (5 cells), 0.06 molarity NaCl
concentration has (6 cells) but the highest number of
telophase observed in 0.12 molarity NaCl concentration
(7 cells).
Cell division is one of the most important
phenomena. Cell division controls organisms growth
and chromosomes behavior is one of the unique
characteristics. The treatment of Allium cepa root tips
with different concentrations of NaCl induced six major
types of chromosomal abnormalities, including
micronuclei, budding nuclei, c-mitosis, unequal-sized
nuclei, anaphase bridge, and chromosome stickiness.
The inhibitory effects of NaCl have previously
been recorded on the root growth of Chrysanthemum
morifolium Ramat, four vegetables (Beta vulgaris,
Brassica oleracea var. capitata L., Amaranthus
paniculatus and
Brassica campestris) and red
raspberry (Hossain et al., 2004; Jamil et al., 2006;
Neocleous & Vasilakakis, 2007). Karyotype analysis
has been carried out extensively in plant phylogenetic
and diversity studies for over 100 years (Ramesh,
2015). Soil salinity is a major abiotic stress in
agricultural plants around the world. Information on the
genetic diversity of crops is important for successful
breeding program development (Barakat, 2003).
In anaphase–telophase cells, the bridge, stickiness,
vagrant chromosomes, fragments, c-anaphase and
multipolarity chromosome aberrations have been
observed (Yildiz et al., 2009). Different cytological
abnormalities were scored, such as break, gap,
exchange, multiple breaks, and chromosome fragments
(Palanikumar et al., 2011). The stick chromosomes
induced abnormal uncoiling of chromosomes during
anaphase to telophase (Qian et al., 2006).
Table 3 and Fig. 3 showed that various
chromosomal abnormalities induced by different NaCl
concentrations in meristematic cells of Allium cepa root
tips. In the control group (distill water) total
chromosomal abnormalities are 0 (0%). The total
chromosomal abnormalities in the 0.48 molarity NaCl
concentration were 21 (51.2%). Micronuclei and
budding nuclei have an approximate half-percentage 9
(21.9%), whereas c-mitosis and chromosome stickiness
have the same proportion 3 (7.3%). Unequal-sized
nuclei aberrations are 5 (12.1%). Anaphase bridge has
the lowest percentage 1 (2.04%). On the other hand,
0.06 molarity NaCl concentration has only 1 (1.9%) of
c-mitosis and chromosome stickiness. But the
remaining aberrations (micronuclei and budding nuclei,
J. Adv. Lab. Res. Biol. (E-ISSN: 0976-7614) - Volume 11│Issue 1│January 2020
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Effects of NaCl on the root tips of Allium cepa
Harem Othman Smail
unequal-sized nuclei and anaphase bridge) have not
occurred. Total abnormalities for 0.06 molarity NaCl
concentration were 2 (3.8%). In 0.12 molarity NaCl
concentration micronuclei and budding nuclei, unequalsized nuclei and c-mitosis have the lowest percentage 1
(1.9%), followed by anaphase bridge 2 (3.9%).
Chromosome stickiness has the highest percentage 3
(5.8%). Total abnormalities for 0.12 molarity NaCl
concentration were 8 (15.6%). In 0.24 molarity NaCl
concentration unequal-sized nuclei and anaphase bridge
has similar percentage 1 (2.04%). Likewise, c-mitosis
and chromosome stickiness have the same number 2
(4.08%). Micronuclei and budding nuclei have the
highest percentage 4 (8.1%). Total abnormalities for
0.24 molarity NaCl concentration were 11 (22.4%).
Table 1: Inhibitory effect of different concentrations of NaCl on Allium cepa root growth.
Concentration
0 (control)
0.06
0.12
0.24
0.48
Mean root length (±SEM) at time (hour)
24
48
72
1.28±0.08
1.42±0.1
1.8±0.8
0.92±0.03
1.05±0.5
1.25±0.5
0.95±0.05
0.96±0.01
1.15±0.05
0.825±0.04
0.85±0.07
0.95±0.05
0.62±0.03
0.5±0.06
0.7±0.04
Change rate (100%) at 72 hr.
40.63%
35.86%
21.05%
15.15%
12.9%
Table 2: Effect of different concentrations of NaCl on the mitotic index of the examined root tip cells of Allium cepa.
Concentration Total cells examined Interphase
450
396
0 (control)
450
398
0.06
450
399
0.12
450
401
0.24
450
409
0.48
Prophase
27
24
31
33
29
Metaphase
14
16
4
4
7
Anaphase
8
6
9
8
2
Telophase
5
6
7
4
3
Mitotic Index (%)
12%
11.55%
11.33%
10.88%
9.11%
Table 3: Chromosome abnormalities induced by NaCl in Allium cepa.
NaCl concentrations
0 (control )
0.06
0.12
0.24
0.48
Micronuclei and Budding
Nuclei (%)
0 (0%)
0 (0%)
1 (1.9%)
4 (8.1%)
9 (21.9%)
Unequal-sized
nuclei (%)
0 (0%)
0 (0%)
1 (1.9%)
1 (2.04%)
5 (12.1%)
c-Mitosis
(%)
0 (0%)
1 (1.9%)
1 (1.9%)
2 (4.08%)
3 (7.3%)
Anaphase
bridge (%)
0 (0%)
0 (0%)
2 (3.9%)
1 (2.04%)
1 (2.4%)
Chromosome
Total abnormal
stickiness (%)
0 (0%)
0 (0%)
1 (1.9%)
2 (3.8%)
3 (5.8%)
8 (15.6%)
2 (4.08%)
11 (22.4%)
3 (7.3%)
21 (51.2%)
Fig. 1: Meristematic cells of root tips of Allium cepa in different state observed at magnification 40X.
(A) - Control (B) - 0.06 molarity of NaCl (C) - 0.12 molarity of NaCl (D) - 0.24 molarity of NaCl (E) - 0.48 molarity of NaCl.
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Effects of NaCl on the root tips of Allium cepa
Harem Othman Smail
Fig. 2: Meristematic cells of root tips of Allium cepa in different stages of cell cycle observed at magnification 100X.
(A) - Interphase (B) - Prophase (C) - Metaphase (D) - Anaphase (E) - Telophase.
Fig. 3: Different types of chromosomal abnormalities induced by NaCl.
(A) - Budding Nuclei (B) - Unequal-sized nuclei (C) - c-mitosis (D) - Anaphase bridge (E) - Chromosome stickiness (F) - Micronucleus.
The p-value was 0.002932 and confirmed that the
different concentrations of NaCl have an effect on the
chromosomal abnormalities and the results were
statistically significant at p-value < 0.05. The p-value
for mean root growth was 0.000682, indicated that the
results were statistically significant. While for mitotic
index p-value was 1, which means the results were
statistically non-significant.
4. Conclusion
The results obtained from root growth
(macroscopic) and cytological analysis (microscopic) of
Allium cepa suggested that different concentrations of
NaCl showed an inhibitory effect on root growth,
decrease cell division and promote chromosomal
abnormalities.
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